Ectodomain shedding of human Nogo-66 receptor homologue-1 by zinc metalloproteinases.

The Nogo-66 receptor (NgR) plays a pivotal role in the inhibition of neuroregeneration as the receptor for multiple neurite outgrowth inhibitors such as Nogo-A. We have previously shown that NgR undergoes zinc metalloproteinase-mediated ectodomain shedding in neuroblastoma cells. Here, we demonstrate that the NgR-related protein NgR homologue-1 is released from neuroblastoma cells as a full-length ectodomain (NgRH1-ecto) and an N-terminal fragment (NTF-NgRH1) containing the leucine-rich repeat region of the protein. Inhibitors of the major protease classes failed to block the release of NgRH1-ecto, suggesting that this occurs via a protease-independent mechanism, presumably by a phospholipase-like enzyme. The release of NTF-NgRH1 was blocked by a hydroxamate-based zinc metalloproteinase inhibitor and tissue inhibitor of metalloproteinases-2 and -3, but not -1, implicating the involvement of membrane-type matrix metalloproteinases in this process. Our findings thus highlight the parallels between the ectodomain shedding of NgRH1 and that previously described for NgR

The selective anti-IL17A monoclonal antibody secukinumab (AIN457) attenuates IL17A-induced levels of IL6 in human astrocytes

The family of interleukin 17 receptors (IL17Rs), subtypes IL17RA-IL17RE, is targeted by the group of pro-inflammatory IL17 cytokines (IL17A-F) and moreover the newly developed anti-IL17A antibody secukinumab (AIN457) has shown promise in Phase II trials in multiple sclerosis. Here, we show that human astrocytes, isolated from a fetal cerebral cortex, express IL17RA and IL17RC and in vitro treatment with IL17A increases protein levels of IL6 in human astrocytes, which is enhanced in the presence of TNFα, as determined by homogeneous time resolved fluorescence. Studies on acutely isolated mouse astrocytes are comparable to human astrocytes although the protein levels of IL6 are lower in mouse astrocytes, which also show a lower response to IL17F and IL1β in promoting IL6 levels. In human astrocytes, IL17A and TNFα also induce mRNA expression of IL6, IL8 and the Th17 cytokines CXCL1, CXCL2, and CCL20, with little effect on Th1 cytokines CXCL9, CXCL10, and CXCL11. The effects of IL17A are associated with nuclear translocation of the NF-κB transcription factor, as determined by immunocytochemistry, where treatment of human astrocytes with the inhibitors of the NF-κB pathway and with secukinumab inhibits the IL17A and IL17A/TNFα-induced increase in nuclear translocation of NF-κB and levels of IL6. Taken together the data shows that IL17A signaling plays a key role in regulating the levels of cytokines, such as IL6, in human astrocytes via a mechanism that involves NF-κB signaling and that selective inhibition of IL17A signaling attenuates levels of pro-inflammatory molecules in astrocytes. © 2014 Wiley Periodicals, Inc

The dual S1PR1/S1PR5 drug BAF312 (Siponimod) attenuates demyelination in organotypic slice cultures

Background: BAF312 (Siponimod) is a dual agonist at the sphingosine-1 phosphate receptors, S1PR1 and S1PR5. This drug is currently undergoing clinical trials for the treatment of secondary progressive multiple sclerosis (MS). Here, we investigated the effects of BAF312 on isolated astrocyte and microglia cultures as well as in slice culture models of demyelination. Methods: Mouse and human astrocytes were treated with S1PR modulators and changes in the levels of pERK, pAkt, and calcium signalling as well as S1PR1 internalization and cytokine levels was investigated using Western blotting, immunochemistry, ELISA and confocal microscopy. Organotypic slice cultures were prepared from the cerebellum of 10-day-old mice and treated with lysophosphatidylcholine (LPC), psychosine and/or S1PR modulators, and changes in myelination states were measured by fluorescence of myelin basic protein and neurofilament H. Results: BAF312 treatment of human and mouse astrocytes activated pERK, pAKT and Ca2+ signalling as well as inducing S1PR1 internalization. Notably, activation of S1PR1 increased pERK and pAKT in mouse astrocytes while both S1PR1 and S1PR3 equally increased pERK and pAKT in human astrocytes, suggesting that the coupling of S1PR1 and S1PR3 to pERK and pAKT differ in mouse and human astrocytes. We also observed that BAF312 moderately attenuated lipopolysaccharide (LPS)- or TNFaα/IL17-induced levels of IL6 in both astrocyte and microglia cell cultures. In organotypic slice cultures, BAF312 reduced LPC-induced levels of IL6 and attenuated LPC-mediated demyelination. We have shown previously that the toxic lipid metabolite psychosine induces demyelination in organotypic slice cultures, without altering the levels of cytokines, such as IL6. Importantly, psychosine-induced demyelination was also attenuated by BAF312. Conclusions: Overall, this study suggests that BAF312 can modulate glial cell function and attenuate demyelination, highlighting this drug as a further potential therapy in demyelinating disorders, beyond MS

Characterization of white matter damage in animal models of multiple sclerosis by magnetization transfer ratio and quantitative mapping of the apparent bound proton fraction f.

Quantitative magnetization transfer magnetic resonance imaging (qMT-MRI) can be used to improve detection of white matter tissue damage in multiple sclerosis (MS) and animal models thereof. To study the correlation between MT parameters and tissue damage, the magnetization transfer ratio (MTR), the parameter f* (closely related to the bound proton fraction) and the bound proton transverse relaxation time T(2B) of lesions in a model of focal experimental autoimmune encephalomyelitis (EAE) were measured on a 7T animal scanner and data were compared with histological markers indicative for demyelination, axonal density, and tissue damage. A clear spatial correspondence was observed between reduced values of MTR and demyelination in this animal model. We observed two different levels of MTR and f* reduction for these lesions. One was characterized by a pronounced demyelination and the other corresponded to a more severe loss of the cellular matrix. Changes in f* were generally more pronounced than those of MTR in areas of demyelination. Moreover, a reduction of f* was already observed for tissue where MTR was virtually normal. No changes in T(2B) were observed for the lesions. We conclude that MTR and qMT mapping are efficient and reliable readouts for studying demyelination in animal models of MS, and that the analysis of regional f* might be even superior to the analysis of MTR values. Therefore, quantitative mapping of f* from human brains might also improve the detection of white matter damage in MS

Zinc metalloproteinase-mediated cleavage of the human Nogo-66 receptor.

The central nervous system myelin components oligodendrocyte-myelin glycoprotein, myelin-associated glycoprotein and the Nogo-66 domain of Nogo-A inhibit neurite outgrowth by binding the neuronal glycosyl-phosphatidylinositol-anchored Nogo-66 receptor (NgR) that transduces the inhibitory signal to the cell interior via a transmembrane co-receptor, p75NTR. Here, we demonstrate that human NgR expressed in human neuroblastoma cells is constitutively cleaved in a post-ER compartment to generate a lipid-raft associated C-terminal fragment that is present on the cell surface and a soluble N-terminal fragment that is released into the medium. Mass spectrometric analysis demonstrated that the N-terminal fragment terminated just after the C-terminus of the ligand-binding domain of NgR. In common with other shedding mechanisms, the release of this fragment was blocked by a hydroxamate-based inhibitor of zinc metalloproteinases, but not by inhibitors of other protease classes and up-regulated by treatment with the cellular cholesterol depleting agent methyl-beta-cyclodextrin. The N-terminal fragment bound Nogo-66 and blocked Nogo-66 binding to cell surface NgR but failed to associate with p75NTR, indicative of a role as a Nogo-66 antagonist. Furthermore, the N- and C-terminal fragments of NgR were detectable in human brain cortex and the N-terminal fragment was also present in human cerebrospinal fluid, demonstrating that NgR proteolysis occurs within the human nervous system. Our findings thus identify a potential cellular mechanism for the regulation of NgR function at the level of the receptor

Design and Synthesis of Selective and Potent Orally Active S1P5 Agonists

Putting the brakes on demyelination: Fingolimod (FTY720) was recently shown to significantly decrease relapse rates in patients with multiple sclerosis. This drug attenuates the trafficking of harmful T-cells entering the brain by regulating sphingosine-1-phosphate (S1P) receptors. We designed, synthesized, evaluated 2H-phthalazin-1-one derivatives (e.g., 1 L) as selective S1P5 receptor agonists; these compounds are highly potent and selective, with good PK properties, and significant activity in oligodendrocytes

The second generation active Aβ immunotherapy CAD106 reduces amyloid accumulation in APP transgenic mice while minimizing potential side effects

Immunization against Aβ can reduce amyloid accumulation in vivo and is considered a potential therapeutic approach for Alzheimer’s disease. However, it has been associated with meningoencephalitis thought to be mediated by inflammatory T cells. With the aim of producing an immunogenic vaccine without this side effect we designed CAD106 comprising Aβ1-6 coupled to the virus-like particle Qβ. Immunization with this vaccine did not activate Aβ-specific T cells. In APP transgenic mice CAD106 induced efficacious Aβ antibody titers of different IgG subclasses mainly recognizing the Aβ3-6 epitope. CAD106 reduced brain amyloid accumulation in two APP transgenic mouse lines. Plaque number was a more sensitive readout than plaque area followed by Aβ42 and Aβ40 levels. Studies with very strong overall amyloid reduction showed an increase in vascular Aβ, which atypically was non-fibrillar. The efficacy of Aβ immunotherapy depended on the Aβ levels and thus differed between animal models, brain regions and stage of amyloid deposition. Therefore, outcomes from animal studies may not be quantitatively applicable to human AD. Our studies provided no evidence for increased microhemorrhages or inflammatory reactions in amyloid-containing brain. In Rhesus monkeys CAD106 induced a similar antibody response as in mice. The antibodies stained amyloid deposits on tissue sections of mouse and human brain but did not label cellular structures containing APP. They reacted with Aβ mono- and oligomers and blocked Aβ toxicity in cell culture. We conclude that CAD106 immunization is suited to interfere with Aβ aggregation and its downstream detrimental effects

ADAM17 is the main sheddase for the generation of human triggering receptor expressed in myeloid cells (hTREM2) ectodomain and cleaves TREM2 after Histidine 157

Triggering receptor expressed in myeloid cells (TREM2) is a member of the immunoglobulin superfamily and is expressed in macrophages, dendritic cells, microglia, and osteoclasts. TREM2 plays a role in phagocytosis, regulates release of cytokine, contributes to microglia maintenance, and its ectodomain is shed from the cell surface. Using both pharmacological and genetic approaches we report here that the main protease contributing to the release of TREM2 ectodomain is ADAM17, (a disintegrin and metalloproteinase domain containing protein, also called TACE, TNFα converting enzyme) while ADAM10 plays a minor role. Using mutational analysis, we demonstrate that the main cleavage site of the sheddases is located within the stalk region of TREM2 proximal to the plasma membrane. Complementary biochemical experiments reveal that cleavage occurs between histidine 157 and serine 158. Shedding is not altered for the R47H-mutated TREM2 protein that confers an increased risk for the development of Alzheimer’s disease. O-glycosylation is detected within the stalk region, but distant to the cleavage site. These findings reveal a link between shedding of TREM2 and its regulation during inflammatory conditions or chronic neurodegenerative disease like AD in which activity or expression of sheddases might be altered